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GST-Ran fusion proteins were purified and loaded with guanine nucleotide as described (16). Each protein was concentrated to 250 μM in ultrafree centrifugal filters (Millipore), then frozen in liquid nitrogen before storage at -80°C GST-Ran fusion proteins were diluted to 25 μM in CSF-arrested Xenopus egg extract for each assay. CSF-arrested Xenopus extracts and demembranated sperm were prepared as described (7, 14). The aster formation assay used was the same as described (24). Samples were viewed with a Nikon Eclipse E800 microscope and images captured with a cooled charge-coupled device (CCD) camera (Princeton Instruments) with the IP labs spectrum P software.
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To quantify the microtubule polymer mass in asters or spindles, we took images with a cooled CCD camera at the same exposure and below saturation level of the camera. Fluorescence intensity of each aster or spindle was measured, and the background was subtracted from the area next to each aster or spindle with the IP labs spectrum P software. Over 30 asters or spindles were measured in each experiment.
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We thank L. Zhang and O. Martin for technical support; J. Gall, C. Wiese, and Q. Guo for critical reading the manuscript; members of the Zheng lab for helpful discussion and support; I. Macara (University of Virginia, Charlottesville) and K. Lounsbury (University of Vermont) for the Ran-GST fusion proteins; A. Merdes (University of Edinburgh) for the antibody to NuMA; and D. Card (University of Utah) for XMAP215 sequence information. Supported by NIH grant (GM55312-01) and a Pew Scholar's Award (Y.Z.).
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